Androgen ablation (castration) is one major approach to treat prostate cancer (PCa), as androgen receptor (AR) signaling is essential for PCa development and growth. Enough evidence supports the notion that AR signaling continues to be active in castration-resistant prostate cancer (CRPC). Consequently, abiraterone and enzalutamide, two androgen signaling inhibitors (ASI) are becoming the major drugs to treat CRPC post- docetaxel. Unfortunately, ASI can only improve the overall patient survival for 2-5 months. Therefore, it is urgent to identify new targets and develop novel approaches to treat ASI-resistant CRPC. Accumulating data suggests that the ?-catenin pathway is a good target for CRPC, because the ?-catenin pathway was identified as one of the top signaling pathways with significant genomic alterations in CRPC and activation of the ?- catenin pathway contributes to elevation of AR signaling. However, how the ?-catenin pathway can be targeted in CRPC is still elusive. The long-term goals of this study are to identify novel and druggable signaling pathways that offer more effective treatment options for patients with ASI-resistant CRPC. The objective is to define the role of polo-like kinase 1 (Plk1) in regulating the ?-catenin pathway, thus AR signaling, and to exploit this unique pathway as a novel therapeutic target for CRPC patients. Axin2, a key regulator of the ?-catenin pathway, was identified as a novel Plk1 substrate. The central hypothesis is that a combination of inhibition of Plk1 with BI2536 and the ?-catenin pathway with IWR1 is a new approach to treat ASI-resistant CRPC. This hypothesis will be tested by pursuing three Specific Aims - (1) to determine whether a combination of inhibition of Plk1 and ?-catenin pathway is a novel approach to treat ASI-resistant CRPC using patient- derived xenografts; (2) to test whether inhibition of Plk1 activates the ?-catenin pathway in genetically engineered mice; and (3) to dissect how Plk1 phosphorylation of Axin2 regulates the ?-catenin pathway and AR signaling. These complementary aims will be accomplished using biochemical analyses of signaling intermediates and employing genetic strategies with PCa mouse models, culture systems and PCa xenograft methodologies. The rationale for the research is that it will be the first to probe the importance of Plk1 to the ?-catenin and AR signaling and to examine whether a combination of inhibition of Plk1 and the ?-catenin pathway is a novel approach to treat ASI-resistant CRPC. This contribution is significant because it will () define the molecular mechanism by which Plk1 regulates the ?-catenin and AR signaling; and (ii) validate Plk1 as a critical therapeutic target to enhance the efficacy of inhibitors that targt the ?-catenin pathway. The research is innovative as it approaches the disease from a novel Plk1 signaling pathway, challenging the traditional view that Plk1 functions solely to regulate mitotic events. These studies are poised to provide a new paradigm for improved patient therapies by identifying the key regulator of the ?-catenin and AR signaling that is critical for generating and maintaining the CRPC phenotype.